Granulysin is a newly discovered cytolytic molecule co-expressed with granzymes and perforin in granules of CTL and NK cells. This molecule is expressed constitutively in NK cells but is expressed 3-5 days after activation of T cells. It is released from cytolytic granule upon stimulation via the TCR. Recombinant granulysin lyses tumor targets and bacteria, but does not lyse RBC. The purpose of this proposal is to better define the mechanism of action of granulysin, its target specificity, and expression in disease. The specific aims are as follows: (1) Characterize the mechanism of action of granulysin, including the lytic activity of truncated or mutated forms of granulysin, formation of pores in membranes, association with lipids, ability to induce apoptosis, localization in target cells, and synergy with other molecules. (2) Better define the clinical relevance of granulysin by: (a) evaluating the specificity of lysis on panels of virally infected, microbial, and tumor target cells; and (b) evaluating expression of granulysin in disease sites, focusing on infection and cancer. (3) Generate a granulysin knock-out (KO) mouse in order to confirm its functional importance in vivo and to better characterize its mechanism of action, target specificity, and role in disease. Together these studies should provide a greater understanding of granulysin function and may permit application of this information to the design of new therapies of immune-mediate diseases.